GDF-8 (Growth Differentiation Factor-8), also called myostatin as a growth controlling factor, which selectively negative regulates skeletal muscle growth, is discovered in 1997 (McPherron et al., Nature, 387:83, 1997). A research team, which discovered myostatin, has announced that two high quality cow breeds due to their high muscular mass and tender meat, i.e., Belgian blue and Piedmontese, have mutation in gene encoding myostatin, which results in muscle development (McPherron and Lee, Proc. Natl. Acad. Sci. USA., 94:12457, 1997), and reported that double-muscle animals of these breeds have average muscle mass increased by 20˜25% based on that of ordinary animals.
Experimentally, myostatin-knockout mice also showed significant increases in skeletal muscle mass, and muscles isolated from myostatin-negative mice were about 2- to 3-fold heavier than muscles isolated from wild mice. It has been reported that knockout mice have about 35% higher total body weight than that of wild mice and myostatin-deficient mice have 80% more muscle fibers than that of normal mice, and the increment of skeletal muscle observed in the knockout mice is caused by abnormal growth of muscle fibers as well as an increase in the number of muscle fibers.
Myostatin as a growth controlling factor, which selectively negative regulates skeletal muscle growth, belongs to TGF-α (transforming growth factor-α) super family, is composed of 375 amino acid precursors, and has the same C-terminal fragments of about 109 amino acid residues in mice, rats, human, swine, fowl and turkey and only 3 amino acid residues in the C-terminal region thereof are not the same in monkeys, cows, and sheep. The C-terminal regions are expected to include physiologically active portion of myostatin. Myostatin has shown a high degree of conservation along evolution among various species, which implies that myostatin is an essential factor in biological muscle control (McPherron and Lee, Proc. Natl. Acad. Sci. USA., 94:12457, 1997).
Myostatin expression is limited to skeletal muscle and it is expressed at low levels in adipose tissue. It seems that myostatin functions as a negative regulator of skeletal muscle growth, but the physiological role of myostatin in an adult individual is not known. Although studies on the physiological role of myostatin have been focused on abnormal growth or its regeneration ability after muscle damage, it is also known that myostatin inhibits adipose tissue growth. However, it has not been known yet whether myostatin acts locally or systemically to regulate animal growth.
Recently, various studies are being conducted on inactivating or inhibiting myostatin exerting the role for negative regulation of skeletal muscle growth. Representative studies thereof include the development of therapeutic agents for treatment of human diseases including muscle-wasting diseases such as muscular dystrophy or muscular atrophy, or muscle loss caused by AIDS, cancer and the like, and an attempt to develop feedstuff additives for growing livestock with high quality meat. Moreover, since, when myostatin was developed as supplement additives for muscle enhancement, it inhibited body fat accumulation due to an increase in the amount of muscle, it is expected to be effective for obesity treatment, and thus, studies thereon are also being conducted.
Two representative studies on a method for inducing muscle growth by inhibiting the function of myostatin protein, are being conducted. One is to discover and use various proteins (follastatin, mutant activin type II receptor, myostatin propeptide, etc.) which inhibit myostatin activity to suppress its function, and the other is to produce antibodies against myostatin polypeptide by animal immunization using myostatin polypeptide, a subsequence thereof, and mutant subsequences.
It was reported that the production of antibodies against myostatin immunogens in vertebrate animals results in a reduction in endogenous myostatin activity, thus showing biological effects such as body weight gain, increased muscle mass, an increase in the number of muscle cells, an increase in muscle cell size, a decrease in the amount of body fat, an increase in muscular strength and the like. However, because the number and type of muscle fibers are genetically programmed during embryogenesis, a decrease in myostatin activity does not lead an increase in the number of muscle fibers in fully grown breeding animals, which may negatively affect meat quality, breed characteristics and fat ratio, but an increase in body weight and growth rate caused by abnormal muscle growth in animals showing a decrease in myostatin activity provides effective methods in the production of beef, pork and poultry meat.
However, most of the studies are conducted by artificially synthesizing myostatin polypeptide or the subsequence thereof, or preparing by isolation and purification after expressing them in Escherichia coli, which results in economic inefficiency and thus it is difficult to apply them to industrial applications.
In livestock industry, various breeding programs to enhance the growth rate of animals by increasing feedstuff efficiency are being developed and improved. Among them, medical approaches include methods of administering antibiotics or antibiotic-like compounds to breeding animals, or administering hormones such as growth hormones to them. However, administration of antibiotics or antibiotic-like compounds to breeding animals is banned, since it can cause a problem of inducing antibiotic cross-resistance. Additionally, in the case of administration of growth hormone to breeding animals, it is disadvantageous in that it costs a lot, short period of treatment should be repeated because of a short half-time of growth hormone, and growth hormone remaining in meat obtained from animals treated therewith may cause health problems in humans. Because of the difficulties in the medical approaches, various breeding programs to enhance the growth rate of animals by increasing feedstuff efficiency, are being continuously developed.
Technology of expressing by attaching a desired protein to the cellular surface of microorganisms is referred to as a cell surface display technology. The cell surface display technology is to express a foreign protein on the cellular surface using the surface protein of microorganisms, such as bacteria or yeasts, as a surface anchoring motif, and is used in a wide range of applications, including the production of recombinant live vaccines, the construction and screening of peptide/antibody libraries, whole cell absorbents and bioconversion catalysts. The application range of this technology is determined depending on what kind of protein is expressed on the cell surface, thus the industrial application potentiality of the cell surface display technology can be said to be significant.
For successful cell surface display, a surface anchoring motif is most important. Selection and development of a motif capable of effectively expressing a foreign protein on the cell surface is the core of this technology. Accordingly, a surface anchoring motif with the following properties should be selected. First, it should have a secretory signal helping the foreign protein to pass through an intracellular membrane, and to reach to the cell surface. Second, it should have a target signal helping the foreign protein to be stably attached to the outer surface of the cell membrane. Third, it should be expressed on the cell surface at large amounts but has little or no effect on the growth of cells. Fourth, it should be stably expressed regardless of the protein size, without causing a change in the three-dimensional structure of the foreign protein. However, a surface anchoring motif satisfying all the above requirements have not yet been developed.
Cell surface anchoring motifs, which have been known and used till now, are broadly classified into four kinds, i.e., outer membrane proteins, lipoproteins, secretory proteins, and surface proteins such as flagella proteins. In the case of gram-negative bacteria, proteins present in the outer cell membrane, such as LamB, PhoE (Charbit et al., J. Immunol., 139:1658, 1987; Agterberg et al., Vaccine, 8:85, 1990) and OmpA, were mainly used. Moreover, lipoproteins, such as TraT (Felici et al., J. Mol. Biol., 222:301, 1991), PAL (peptidoglycan associated lipoprotein) (Fuchs et al., Bio/Technology, 9:1369, 1991) and Lpp (Francisco et al., Proc. Natl. Acad. Sci. USA., 489:2713, 1992), were also used. Furthermore, the expression of foreign proteins was also attempted using FimA, a fimbriae protein such as the FimH adhesin of type 1 fimbriae (Hedegaard et al., Gene, 85:115, 1989), or a pili protein such as a PapA pilus subunit as surface anchoring motifs. In addition, it was reported that an ice nucleation protein (Jung et al., Nat. Biotechnol., 16:576, 1998; Jung et al., Enzyme Microb. Technol., 22:348, 1998; Lee et al., Nat. Biotechnol., 18:645, 2000), pullulanase of Klebsiella oxytoca (Kornacker et al., Mol. Microbiol., 4:1101, 1990), IgA protease of Neisseria (Klauser et al., EMBO J., 9:1991, 1990), E. coli adhesin AIDA-1, VirG protein of Shigella, a fusion protein of Lpp and OmpA, can be used as surface anchoring motifs.
In the case of using Gram-positive bacteria, there is a report that a malaria antigen was effectively expressed using Staphylococcus aureus-derived protein A and FnBPB protein, as surface anchoring motifs. In addition, it was reported that surface coat protein of lactic acid bacteria was used in surface expression and surface protein of Gram-positive bacteria such as a Streptococcus pyogenes-derived M6 protein (Medaglini, D et al., Proc. Natl. Acad. Sci. USA., 92:6868, 1995), S-layer protein EA1 of Bacillus anthracis, Bacillus subtilis CotB, etc., were also used as surface anchoring motifs.
Meanwhile, the present inventors already developed a novel vector effectively expressing a foreign protein on the surface of microorganisms using a pgsBCA gene encoding a Bacillus sp. strain-derived poly-gamma-glutamate synthetase complex as a new surface anchoring motif, as well as a method for expressing a large amount of foreign protein on the surface of microorganisms transformed with the vector (Korea Patent Registration No. 10-0469800).
Many studies were performed in an attempt to stably express the antigen or epitope of pathogenic organisms in bacteria where mass production is possible by genetic engineering techniques using the above-described surface anchoring motifs. It was reported that, particularly when a foreign immunogen expressed on the surface of non-pathogenic bacteria is orally administered alive, a more lasting and strong immune response than that of the prior vaccine using attenuated pathogenic bacteria or viruses, can be induced. This induction of the immune reaction is induced by an in vivo immune response to the live bacteria, which is known to be because the surface structures of the bacteria act as adjuvants increasing the antigenicity of the surface-expressed foreign protein. The development of a recombinant live vaccine of non-pathogenic bacteria using this surface expression system is noticeable.
Accordingly, the present inventors have made extensive efforts to develop a method for effectively expressing a myostatin fusion protein, capable of inducing a strong immunogenicity against myostatin, on the surface of a microorganism, and as a result, they found that, when a fusion protein, in which a multimer of Myo-2 peptide is fused to myostatin, was expressed on the surface of lactic acid bacteria using a pgsBCA gene encoding Bacillus sp. strain-derived poly-gamma-glutamate synthetase complex as a surface anchoring motif, said fusion protein was effectively expressed on the surface thereof, and confirmed that oral administration of lactic acid bacteria expressing said fusion protein on the cell surface increased antibody production in blood, as well as, body weight and muscle mass, thereby completing the present invention.